Abstract

About one third of the known cellular oncogenes are transcription factors (TFs). As many tumors are dependent on continuous activity of these proteins they represent highly interesting targets for therapeutic approaches e.g. by inhibition with small molecule inhibitors. Because of lack of enzymatic activity and accompanied difficulty of direct targeting, TF have been considered undruggable for a long time. This view recently changed with the demonstration that, instead of direct targeting, indirect targeting of protein partners required for TF activity might represent an appropriate alternative. Among transcription factors targeted by this approach are Notch, Bcl6 and EWS/FLI, the oncogenic fusion protein in Ewing sarcoma.

In our studies we focus on alveolar Rhabdomyosarcoma (aRMS), an aggressive and highly metastatic pediatric sarcoma. ARMS is characterised by the expression of the fusion TF PAX3/FOXO1 which is essential for the survival of aRMS cells. Here, we aimed to identify interacting proteins that are involved in modulating the aberrant activity of PAX3/FOXO1 in aRMS.

Towards this end, we purified the interactome of PAX3/FOXO1 from two different aRMS cell lines and identified the individual interactors by mass spectrometry. This led to the description of 150 candidate interactors which were reproducibly co-purified with PAX3/FOXO1 in four independent experiments. Most of the identified proteins are transcription factors or transcriptional regulators. To validate our findings, selected candidate interactors such as RUNX1 and TFAP2B were further shown to pull down PAX3/FOXO1 in co-immunoprecipitation studies.

To analyse whether some of the candidate interactors have an influence on PAX3/FOXO1 activity we individually silenced the expression of a set of 60 candidates by siRNA and measured the expression of several PAX3/FOXO1 target genes including TFAP2B, FGFR4, and NMYC. These experiments revealed that interactors can have different effects on specific PAX3/FOXO1 target genes and suggest that specific transcriptional complexes are responsible for activating the transcription of different PAX3/FOXO1 target genes. This potentially reflects the diversity of mechanisms by which PAX3/FOXO1 regulates target gene expression including enhancer- and promoter-directed actions. Further characterization of selected interactors is necessary to understand the impact of these on the oncogenic function of PAX3/FOXO1 in more detail.

The results of these experiments shed light on the regulatory pathway(s) and mechanisms involved in PAX3/FOXO1 mediated oncogenesis and thereby contribute to our understanding of the oncogenic function of tumor-specific chimaeric transcription factors.